功能模塊的集成與測(cè)試研究

劉雙江　劉一鳴　劉暢　張博

摘 要：谷氨酸棒桿菌作為棒桿菌中的模式生物，擁有多條完整的芳香化合物代謝途徑，全基因組測(cè)序的完成為在谷氨酸棒桿菌中進(jìn)行代謝調(diào)控研究提供了良好的生物信息學(xué)平臺(tái)；該菌包括基因敲除及互補(bǔ)在內(nèi)的遺傳操作系統(tǒng)非常成熟，是研究芳香化合物代謝調(diào)控機(jī)制的良好模型。該研究旨在利用棒桿菌等模式生物中的莽草酸合成及芳香化合物代謝相關(guān)元件進(jìn)行元件適配性研究，同時(shí)結(jié)合生物信息學(xué)、分子生物學(xué)及生物化學(xué)方法發(fā)掘其他微生物中這兩類元件，并對(duì)元件進(jìn)行表征、改造及標(biāo)準(zhǔn)化并建立元件庫(kù)；選取高效能的功能元件拼接組裝為功能模塊，并在棒桿菌等底盤細(xì)胞中進(jìn)行檢測(cè)適配性，從而構(gòu)建出高效合成以莽草酸為代表性芳香化合物的人工細(xì)胞。到目前為止，研究工作完成了谷氨酸棒桿菌莽草酸合成途徑酶元件的鑒定，重點(diǎn)完成了谷棒DAHP合酶和分支酸異構(gòu)酶功能表征及元件間適配性關(guān)系，獲得大量潛在的莽草酸合成相關(guān)代謝元件；并對(duì)部分代謝元件進(jìn)行功能驗(yàn)證和表征；同時(shí)建立高效蛋白表達(dá)及酶活測(cè)定體系。鑒定了谷棒莽草酸途徑的7個(gè)酶以及分支酸異構(gòu)酶，完成了谷棒DAHP合酶、分支酸異構(gòu)酶、脫氫奎尼酸脫水酶以及莽草酸激酶功能表征，揭示了DAHP合酶和分支酸異構(gòu)酶相互作用機(jī)理和調(diào)控關(guān)系。獲得了3 549個(gè)莽草酸途徑相關(guān)基因序列，確定了516個(gè)合成目標(biāo)基因，完成了這些基因密碼子優(yōu)化和基因序列重新設(shè)計(jì)，選取了37個(gè)脫氫奎尼酸脫水酶基因人工合成，構(gòu)建標(biāo)準(zhǔn)元件庫(kù)，并表征了它們的酶促動(dòng)力學(xué)參數(shù)。構(gòu)建并驗(yàn)證了快速高通量的篩選—優(yōu)化—合成—表征莽草酸途徑元件庫(kù)的Pipeline。另外在調(diào)控元件庫(kù)構(gòu)建方面，構(gòu)建了包括RBS、Promoter、Terminator以及Insulator等4類共226個(gè)元件的調(diào)控元件庫(kù)，為莽草酸通路模塊的優(yōu)化和精細(xì)調(diào)控的奠定了基礎(chǔ)。 模塊的組裝和優(yōu)化方面，構(gòu)建了基于RiboJ Insulator的基因表達(dá)定量調(diào)控模型，合成了莽草酸本地途徑酶元件和調(diào)控元件元件進(jìn)行模塊組裝，并在底盤細(xì)胞谷氨酸棒桿菌中實(shí)現(xiàn)了表達(dá)，對(duì)最優(yōu)配比進(jìn)行了初步篩選，將莽草酸產(chǎn)量提高了7倍。

關(guān)鍵詞：莽草酸 莽草酸途徑 谷氨酸棒桿菌 合成生物學(xué) 元件庫(kù)

Abstract： Corynebacterium glutamicum as a type strain has a number of complete metabolic pathways of aromatic compounds. The completion of whole genome sequencing provides a good bioinformatic platform for metabolic and regulatory study of cells. Besides， the genetic manipulation systems （including knockout and complementary systems） are very mature， which makes this strain a perfect model to study the metabolic and regulatory mechanisms of aromatic compounds.With techniques of bioinformatics， molecular biology and biochemistry， more devices with similar functions are explored from all other microorganisms. And device libraries are subsequently established after characterization， modification and standardization of these devices. High-performance functional devices are selected to assemble modules which are then transferred into chassis cells for suitability test. After optimization of the suitability， artificially synthesized cell can provide a much more efficient synthesis of shikimic acid -a representation of aromatic compounds- than the wild type strain do. So for， all enzymatic devices involved in shikimic acid synthetic pathway have been identified， and a lot of potential function devices that may related to shikimic acid synthesis have been achieved. In total， 3549 gene sequences that are relative to shikimate pathway are identified and 516 of them are verified to be target genes for shikimic acid synthesis. After codon optimization and sequence redesign， 37 dehydrogenation quinic acid dehydratase genes are selected to be synthesis chemically， and these synthetic devices are used for characterization of their enzymatic kinetic parameters. By then， a highly efficient pipeline for construction of device library with a high-throughput “Screening—Optimization—Synthesis—Characterization” process is built. In terms of regulatory devices， a library is constructed with 226 regulatory devices， including RBS， Promoter， Terminator and Insulator.， which provide a steady foundation for optimization and accurate regulation of shikimic acid pathway modules. Based on a quantitative model named RiboJ Insulator for regulation of gene expression， the local enzymes in shikimic acid pathway are assembled with regulatory devices from the previous library in chassis cells Corynebacterium glutamicum. And the production of shikimic acid is increased by 7 times comparing with the wild type ones.

Key Words： Shikimic acid； Shikimic acid pathway； Corynebacterium glutamicum； Synthetic biology； Device library

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